THE POTENTIAL SYNERGISTIC EFFECTS OF pH AND DISINFECTANTS ON INACTIVATION OF PSEUDOMONAS FLUORESCENS ATCC 13525: DISRUPTION OF CELLULAR REDOX HOMEOSTASIS AND ULTRASTRUCTURE

The aim of thesis was to understand the alterations in cellular oxidative processes, antioxidant defense mechanisms, cell ultrastructure and morphology underlying the synergistic bacterial control effects of selected pH values alone (pH 8.2 and 5.0) and peracetic acid, H2O2, dodecyltrimethylammonium chloride DTACl (cationic surfactant), C10 linear alkyl chain alcohol ethoxylate C10/EO8 (non-ionic surfactant) and novel combination of hydrogen peroxide + peracetic acid + non-ionic surfactant with bleach activator sodium nonanoyloxybenzenesulfonate (NOBS) at mild alkaline conditions (pH 8.2), and of linear alkylbenzene sulfonate LAS (anionic surfactant) at acidic milieu (pH 5.0) in Pseudomonas fluorescens ATCC 13525 by measuring the intracellular antioxidant molecules and oxidative stress biomarkers levels, antioxidant enzymes activities, and characterizing the cell morphology and ultrastructure. The minimum inhibitory concentrations (MICs) of peracetic acid, hydrogen peroxide, dodecyltrimethylammonium chloride, linear alkyl chain alcohol ethoxylate and combination of hydrogen peroxide + peracetic acid + non-ionic surfactant with bleach activator at pH 8.2, and MIC of linear alkylbenzene sulfonate at pH 5.0 were used during the stationary growth phase of planktonic cells grown in tryptic soy broth liquid medium to test free radicals generation, catalase, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PDH) specific activities, malondialdehyde (lipid peroxide), protein carbonyls (protein oxidation product), total thiols and low molecular weight thiols (LMWT) levels, and total glutathione/glutathione disulfide (tGSH/GSSG) ratio in addition to analysis of cell ultrastructure and morphology. Hydrogen peroxide and peracetic acid decomposition kinetics by the selected bacterial strain were also investigated. The analyses were performed by UV/VIS spectrophotometer and electron paramagnetic resonance (EPR), and cellular visualization was carried out by transmission electron microscope (TEM). Alkaline conditions (pH 8.2) alone mainly elevated protein carbonyls level, decreased the levels of LMWT and tGSH/GSSG ratio which could potentiate the effect of hydrogen peroxide, peracetic acid, cationic and non-ionic surfactants and selected combination, while acidic environment (pH 5.0) alone mainly decreased total thiol level that could facilitate anionic surfactant action. Free radicals have been detected in highly diluted samples of anionic surfactant and peracetic acid only (10 μg/ml total soluble protein), which explain the higher efficacy of anionic surfactant and peracetic acid when compared to tested disinfectants. Hydrogen peroxide and peracetic acid increased the levels of malondialdehyde and protein carbonyls, while reduced the total thiols and LMWT levels which could explain the oxidative damage of these oxidants to cells. Hydrogen peroxide and peracetic acid were also found to stimulate SOD specific activity and inhibit catalase specific activity which suggest a late stage of oxidative stress. Peracetic acid and hydrogen peroxide increased GR, GPx and G6PDH specific activities which suggests disruption of cellular redox status as a result of oxidation. Cationic surfactant targeted the cell membrane, which was reflected by elevation in malondialdehyde levels. SOD and catalase specific activities were increased, while GPx and G6PDH specific activities were decreased with cationic surfactant. The decrease of GR specific activity also contributed to the effect of cationic surfactant. Non-ionic surfactant caused protein denaturation reflected by increment in total thiol level which is the main target of non-ionic surfactant. Moreover, non-ionic surfactant was found to increase SOD specific activity and decrease the catalase specific activity which suggest cell oxidative damage. Non-ionic surfactant stimulated GR and G6PDH specific activities, while GPx specific activity was not changed suggesting overall imbalance of cell homeostasis. Combination of hydrogen peroxide + peracetic acid + non-ionic surfactant + bleach activator reduced the tGSH/GSSG ratio that explains the bacterial control effects of combination. The same combination was also found to increase GR specific activity, and lower both GPx and G6PDH specific activities as a consequence of oxidative stress. Anionic surfactant increased the levels of protein carbonyls which is a biomarker of protein oxidation, while decreased the total thiol level and tGSH/GSSG ratio. Anionic surfactant was also found to increase SOD specific activity, decrease catalase specific activity, inhibit GR specific activity, while found to stimulate GPx and G6PDH specific activities which altogether impairs the redox status of cell. Each treatment has shown distinct alterations and disruption in cell morphology and cytoplasmic structures as reported by TEM, which result in bacterial control effects. Moreover, the selected bacterial strain decomposed hydrogen peroxide by catalase and peroxidase activities, but did not degrade peracetic acid, due to the lack of cellular enzymes that break down peracetic acid which explains the higher efficacy of peracetic acid in respect of hydrogen peroxide. In conclusion, we suggest that treating bacteria with the tested oxidizing agents and surfactants at the selected pH values probably caused a high production of free radicals which developed intricate oxidative processes and antioxidant defense mechanisms causing an imbalance of the cell redox homeostasis that resulted in deformity and collapse of cellular ultrastructure and morphology at late stage of oxidative damage that led to controlling the growth of Pseudomonas fluorescens. In addition, hydrogen peroxide + peracetic acid + non-ionic surfactant + bleach activator tested combination was found to provide synergistic action for enhanced removal of biological soil and control of bacterial growth.